Selective Modulation of DNA Polymerase Activity by Fixed‐Conformation Nucleoside Analogues

Eoff, Robert L.; McGrath, Colleen E.; Maddukuri, Leena; Salamanca-Pinzón, S. Giovanna; Márquez, Víctor E.; Marnett, Lawrence J.; Guengerich, F. Peter; Egli, Martin

doi:10.1002/anie.201003168

Cited by 16 publications

(22 citation statements)

References 46 publications

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“…Under these conditions, Dpo4 catalysis was slowed nearly 9-fold by S-MC-dATP when compared to the RNA-like pucker of N-MC-dATP. These results are similar to those obtained previously with HIV-1 Reverse Transcriptase (HIV-1 RT) (21) and for human DNA polymerase kappa (hpol κ) (22). In contrast to Dpo4, the B-family member, Dpo1, can catalyze insertion of both N-MC-dATP and S-MC-dATP opposite template dT with relatively modest levels of inhibition by either nucleotide analogue (Figure 1D).…”

Section: Discussionsupporting

confidence: 90%

Differential Furanose Selection in the Active Sites of Archaeal DNA Polymerases Probed by Fixed-Conformation Nucleotide Analogues

et al. 2012

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DNA polymerases select for the incorporation of deoxyribonucleotide triphosphates (dNTPs) using amino acid side-chains that act as a “steric-gate” to bar improper incorporation of rNTPs. An additional factor in the selection of nucleotide substrates resides in the preferred geometry for the furanose moiety of the incoming nucleotide triphosphate. We have probed the role of sugar geometry during nucleotide selection by model DNA polymerases from Sulfolobus solfataricus using fixed conformation nucleotide analogues. North-methanocarba-dATP (N-MC-dATP) locks the central ring into a RNA-type (C2′-exo, North) conformation near a C3′-endo pucker and South-methanocarba-dATP (S-MC-dATP) locks the central ring system into a (C3′-exo, South) conformation near a C2′-endo pucker. Dpo4 preferentially inserts N-MC-dATP and in the crystal structure of Dpo4 in complex with N-MC-dAMP, the nucleotide analogue superimposes almost perfectly with Dpo4 bound to unmodified dATP. Biochemical assays indicate that the S. solfataricus B-family DNA polymerase Dpo1 can insert and extend from both N-MC-dATP and S-MC-dATP. In this respect, Dpo1 is unexpectedly more tolerant of substrate conformation than Dpo4. The crystal structure of Dpo4 bound to S-MC-dADP shows that poor incorporation of the Southern pucker by the Y-family polymerase results from a hydrogen bond between the 3′-OH group of the nucleotide analogue and the OH group of the steric gate residue, Tyr12, shifting the S-MC-dADP molecule away from the dNTP binding pocket and distorting the base pair at the primer-template junction. These results provide insights into substrate specificity of DNA polymerases, as well as molecular mechanisms that act as a barrier against insertion of rNTPs.

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Section: Discussionsupporting

confidence: 90%

Differential Furanose Selection in the Active Sites of Archaeal DNA Polymerases Probed by Fixed-Conformation Nucleotide Analogues

et al. 2012

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“…Yet, it is this feature that appears to explain the increased catalytic efficiency of hpol ι. The stabilized anti conformation results in faster insertion than unmodified dATP, as evidenced by an increased turnover number for N-MC-dATP 37 and also improves the accuracy of hpol ι DNA synthesis opposite dT by increasing the propensity of the enzyme to pair adenine with thymidine. Interestingly, the crystal structure of hpol ι inserting dGTP opposite dT reveals that the incoming dGTP resides in a “Western” type O4′-exo pucker ( P = 271.2 °).…”

Section: Resultsmentioning

confidence: 99%

“…41 Previous work has examined the conformational preferences of the furanose ring system for different enzyme classes, including three human Y-family DNA polymerases. 37,48 A general conclusion was that polymerases tend to favor C3′-endo, North puckers over C2′-end, South puckers for the incoming dNTP. However, insertion of MC-dNTPs tends to diminish catalytic efficiencies for most polymerases tested.…”

Section: Discussionmentioning

confidence: 99%

A Nucleotide-Analogue-Induced Gain of Function Corrects the Error-Prone Nature of Human DNA Polymerase iota

et al. 2012

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Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2′-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2′-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle (χ), which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase.

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“…However, the final biological activity of the nucleosides prodrugs depends on their transport across cell membranes, activation to 5 -mono-, di-, or triphosphate anabolites by viral and/or cellular kinases and their ability to serve as substrates for specific DNA polymerase. It has been reported that different DNA polymerases, including those within a subfamily, show important differences in preferences for the furanose conformation of the sugar moiety during catalysis [12]. The preference of human nucleoside transporters with respect to the sugar ring has been examined using methanocarba-Ado or methanocarba-Cyd in North (N-MCdA and N-MCdC) or South (S-MCdA and S-MCdC) analogs [13].…”

Section: Introductionmentioning

confidence: 99%

Selective Phosphorylation of South and North-Cytidine and Adenosine Methanocarba-Nucleosides by Human Nucleoside and Nucleotide Kinases Correlates with Their Growth Inhibitory Effects on Cultured Cells

Sjuvarsson

Márquez

Eriksson

2015

Nucleosides, Nucleotides and Nucleic Acids

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Here bicyclo[3.1.0]hexane locked deoxycytidine (S-MCdC, N-MCdC), and deoxyadenosine analogs (S-MCdA and N-MCdA) were examined as substrates for purified preparations of human deoxynucleoside kinases: dCK, dGK, TK2, TK1, the ribonucleoside kinase UCK2, two NMP kinases (CMPK1, TMPK) and a NDP kinase. dCK can be important for the first step of phosphorylation of S-MCdC in cells, but S-MCdCMP was not a substrate for CMPK1, TMPK, or NDPK. dCK and dGK had a preference for the S-MCdA whereas N-MCdA was not a substrate for dCK, TK1, UCK2, TK2, dGK nucleoside kinases. The cell growth experiments suggested that N-MCdC and S-MCdA could be activated in cells by cellular kinases so that a triphosphate metabolite was formed. List of abbreviations: ddC, 2', 3'-didioxycytosine, Zalcitabine; 3TC, β-L-(-)-2',3'-dideoxy-3'-thiacytidine, Lamivudine; CdA, 2-cloro-2'-deoxyadenosine, Cladribine; AraA, 9-β-D-arabinofuranosyladenine; hCNT 1-3, human Concentrative Nucleoside Transporter type 1, 2 and 3; hENT 1-4, human Equilibrative Nucleoside Transporter type 1, 2, 3, and 4.

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Selective Modulation of DNA Polymerase Activity by Fixed‐Conformation Nucleoside Analogues

Cited by 16 publications

References 46 publications

Differential Furanose Selection in the Active Sites of Archaeal DNA Polymerases Probed by Fixed-Conformation Nucleotide Analogues

Differential Furanose Selection in the Active Sites of Archaeal DNA Polymerases Probed by Fixed-Conformation Nucleotide Analogues

A Nucleotide-Analogue-Induced Gain of Function Corrects the Error-Prone Nature of Human DNA Polymerase iota

Selective Phosphorylation of South and North-Cytidine and Adenosine Methanocarba-Nucleosides by Human Nucleoside and Nucleotide Kinases Correlates with Their Growth Inhibitory Effects on Cultured Cells

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